Acetaminophen is a widely used analgesic. It is available without a prescription and is often used when aspirin may present problems to a patient. At therapeutic doses, serum concentration is usually below 50 mg/L. Toxicity is usually observed if the serum concentration four hours after ingestion of the drug is greater than 300 mg/L. One effect of overdose is liver toxicity. The need for an accurate method of determining the concentration of acetaminophen in serum is therefore apparent.
Known methods of assaying anilides such as acetaminophen utilize arylacylamidase (E.C.3.5.1.13) and an oxidizing agent. Arylacylamidase cleaves the amide bond of the anilide to produce acetate and an aniline such as p-aminophenol. The aniline is then made to react with with a color-forming compound like phenol in the presence of an oxidizing agent such as permanganate or the metal salts of copper or iron to form a color compound like indophenol which can be detected at 615 nm. Other methods use oxidizing agents like periodate, persulfates, or peroxidase. U.S. Pat. Nos. 4,999,288 and 4,430,433 are typical.
One disadvantage of these methods is that metal salts oxidize the aniline very slowly unless they are at alkaline pH, a condition that inactivates arylacylamidase.
U.S. Pat. No. 4,675,290 (issued Jun. 23, 1987, to Matsumoto) discloses a method of assaying peptidase enzyme activity which comprises treating a synthetic dibrominated amide (as substrate) with a sample containing peptidase thereby liberating a dibrominated aniline; oxidizing the thus-liberated dibrominated aniline with an oxidase (e.g. ascorbate oxidase) which consumes oxygen and forms pigment by oxidative condensation of said aniline in the presence of a coupler of a defined formula. This reaction takes place in solution.
Matsumoto's method uses an enzyme and might therefore overcome the problems encountered with the inorganic oxidizing agents of the prior art. However, Matsumoto employs a synthetic dibrominated substrate and is therefore not directly applicable to a test for acetaminophen. There is evidence in the art that in the case of oxidative coupling of phenols for color formation, color yield can be increased when certain halogenophenols are used in the color-forming reaction. However acetominophen as it appears in biological fluids to be tested is not halogenated and is therefore chemically distinguishable from the dibrominated synthetic substrate used in Matsumoto's method.
Further, Matsumoto disclosed a solution assay. An alternative and more convenient assay involves "dry" chemistry, a term that refers to methods and techniques that are carried out using chemical reagents contained in various "dry-to-the-touch" test elements such as "dip and read" test strips, multilayer test elements and the like. "Dry" methods require no liquid for reconstitution or analysis other than the test sample.
Reagents employed in solution assays often do not perform well when adapted to a dry format. Dry elements utilize minute amounts of reagents and test samples and so in a dry element for acetaminophen assay, it is important to employ reagents that give a clear and precise color signal when aniline is oxidatively coupled to a coupler to provide color.
The range of compounds that could be chosen as potential couplers is very wide. Matsumoto neither suggests nor gives guidance to those color forming oxidative couplers that would perform well in dry chemistry. The problem is that the selection of a suitable oxidative coupler for a dry element is essentially empirical.
It would be desirable to have a dry assay for determining acetominophen in biological fluids comprising enzymes and other reagents which remain active when combined and coated in dry format and which give a clear and precise signal on oxidative coupling with a suitable coupler.